CN1479099A - Recombination antigen for tsutsugamushi disease diagnosis and test paper for its rapid diagnosis - Google Patents

Recombination antigen for tsutsugamushi disease diagnosis and test paper for its rapid diagnosis Download PDF

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Publication number
CN1479099A
CN1479099A CNA031473156A CN03147315A CN1479099A CN 1479099 A CN1479099 A CN 1479099A CN A031473156 A CNA031473156 A CN A031473156A CN 03147315 A CN03147315 A CN 03147315A CN 1479099 A CN1479099 A CN 1479099A
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China
Prior art keywords
yochubio
diagnosis
recombinant
antigen
recombinant antigen
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CNA031473156A
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Chinese (zh)
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严延生
邓艳琴
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FUJIAN SANITARY AND ANTIEPIDEMIC SATATION
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FUJIAN SANITARY AND ANTIEPIDEMIC SATATION
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

A recombinant antigen for diagnosing tsutsugamushi disease is prepared through cloning the structural gene of the immunodominance antigen of Rickettsia orientalis, expressing in colibacillus to obtain a great deal of recombinant antigens, and purifying. A high-speed diagnosing test paper is also disclosed.

Description

Be used for the recombinant antigen of yochubio diagnosis and the test paper of quick diagnosis thereof
Technical field
The present invention relates to the antigen of medical diagnosis on disease, is a kind of recombinant antigen of yochubio diagnosis and test paper of quick diagnosis thereof of being used for specifically.
Background technology
Yochubio, claim scrub typhus (scrub typhus) again, be by yochubio east body (Orientiatsutsugamushi, what Ot) cause is the febrile disease of principal character with heating, fash, insect bite ulcer (eschar) and the enlargement of superficial lymph knot, hepatosplenomegaly, ascites appear in severe patient, give treatment to the untimely death that causes.Yochubio mainly is popular in the Asia-pacific region, and promptly from the east of New Guinea, to Afghanistan, south gets New Zealand and Australian northern coastland, north is to Japan, Russian Far East coastal region.China has 23 province's reports to find yochubio, and there is the trend of expansion in the yochubio epidemic-stricken area in recent years.
In the clinical symptoms of yochubio, insect bite ulcer is its specific sign, but mostly the position is hidden owing to insect bite ulcer, and quantity is few, and the report various places of its recall rate are differed greatly, and therefore must be aided with laboratory examination so that in time make a definite diagnosis.Yochubio east body is the strict entozoic microorganism of special sexual cell, and breeding is slow, and purifying is loaded down with trivial details, thereby is difficult to obtain the yochubio east isoantigen of large-scale purification, has limited the development of the method for inspection.Traditional diagnostic method, as outer striking coomb's test Coomb (Weil-Felix test), immunoperoxidase test (IIP), indirect immunofluorescence antibody detection (IFAT) etc., all based on serological reaction, but limited their use because of limitation separately.Though outer striking coomb's test Coomb is simple to operate, diagnosis lacks susceptibility and specificity to yochubio; IFAT has good susceptibility and specificity, but needs fluorescent microscope and well-trained professional, is difficult in the rural area, basic hospital and on-the-spot the use; Though IIP has replaced fluorescent microscope or even do not needed microscope with ordinary optical microscope, but still need to cultivate and purifying yochubio east body, need expensive tissue culturing equipment.Therefore, be badly in need of setting up a kind of easy, quick serological diagnostic method that also can overcome above-mentioned limitation, to satisfy the needs of yochubio east body clinical detection.
(Scrub typhus antigen Sta56) as the abundantest outer membrane protein of yochubio east surface content, can account for 10～15% of bacterial protein to yochubio east body 56kD antigen; Immunogenicity is strong, is the most often discerned by host immune system; Sta56 can react with strain specific monoclonal antibody and group specificity monoclonal antibody, show that Sta56 exists strain specificity epitope and group specificity epi-position, thereby Sta56 is one of main candidate of yochubio diagnostic antigen.
Collaurum is a kind of suspended particle that is in the semi-reduction state, rapid big molecule such as adsorbed proteins and do not influence its activity, collaurum has the red in aubergine of easy resolution, with after big molecule combines, be easy to the naked eye discern, not needing secondary to amplify can the Direct observation result, colloidal gold diagnosis product thereby can directly enter in the clinic even family that does not have complex detection equipment.In recent years, detecting test paper with early pregnancy is that the colloid gold immune diagnostic reagent of representative has entered huge numbers of families, brings great convenience for people's life.
From comprising that interrelated data retrievals such as Chinese patent show, the recombinant antigen that does not have the gene engineering method utilized to obtain at present as yet is used for the diagnosis of yochubio and the relevant report of yochubio fast diagnose test paper.
Summary of the invention
For the recombinant antigen that overcomes prior art and do not have the gene engineering method utilized to obtain as yet is used for the diagnosis of yochubio, the present invention proposes the recombinant antigen that is used for the yochubio diagnosis and the test paper of quick diagnosis thereof.
The technical solution adopted for the present invention to solve the technical problems is:
One. the solution formula that the present invention relates to is:
(1) recombinant antigen lysate: SDS 0.1%
Glycocoll 250mmol/L
Tris.Cl 25mmol/L
(2) colloidal gold solution: 3～5/0,000 tetra chlorauric acid adds 1～2% trisodium citrate and is prepared into colloid gold particle;
(3) two is anti-: anti-human IgG, anti-people IgM resist more, available from Sigma company;
(4) bag is cushioned liquid: 1～3% sucrose in PBS;
(5) basic damping fluid: available from Xiamen Bo Sheng Bioisystech Co., Ltd;
(6) reaction buffer: 0.01～0.05%NaN 3In PBS;
(7) PBS: dissolving 8g NaCl, 0.2g KCl, 1.44g Na in 800ml distilled water 2HPO 4And 0.24gKH 2PO 4, pH value to 7.2～7.6 with hydrochloric acid conditioning solution add water and are settled to 1L.
Two. the technological process of recombinant antigen of the present invention:
(1) from the body Karp pnca gene group of yochubio east, amplifies sta56 gene ORF or big fragment wherein, use the TA clone technology this large dna fragment cloning;
(2) be template with this recombinant plasmid, amplify the sta56 fragment of brachymemma, directed pET30a carrier, transformed into escherichia coli BL21 (DE3), the IPTG abduction delivering of inserting; Western blot confirms that this recombinant protein can be discerned by yochubio patient positive serum;
(3) adopt electroelution method purification of recombinant proteins: after the thalline of collecting when inducing in a large number carries out Ultrasonic Pulverization, ultrasonic supernatant is prepared electrophoresis, determine the position of target stripe in gel, downcut the gel glue that contains target protein and place bag filter to carry out electroelution; Purification of samples carries out SDS-PAGE, determines purity and carries out protein quantification.
Three. test paper manufacture craft of the present invention
(1) with colloid gold label recombinant antigen and mouse IgG;
(2) two kinds of gold mark antigenic solutions mix fully sized glass fibres paper of back, put the freeze dryer freeze-drying;
(3) goat-anti people μ chain 1.0～5.0mg/ml, goat anti-human igg 1.0～5.0mg/ml and sheep anti-mouse igg monoclonal antibody 1.0～5.0mg/ml bag is dried naturally by the NC film;
(4) thieving paper, NC film and the glass fibre that adsorbed golden mark antigen are assembled into detect IgM, IgG and total Ig test strips.
Just the clinical diagnosis of yochubio can be used for the recombinant antigen of the present invention's making and the test strips of utilizing recombinant antigen to be made into.
The invention has the beneficial effects as follows: the present invention utilizes technique for gene engineering, the structural gene of the immunodominance antigen of clone's yochubio east body, and,, promptly can be used for immunology diagnosis behind the purifying to obtain a large amount of cheap recombinant antigens easily at e. coli expression.The recombinant antigen that obtains of method and the fast diagnose test paper limitation that overcome existing yochubio diagnostic method thus, can be fast, diagnosis of tsutsugamushi disease easily, need not specific apparatus, need not well-trained professional, can satisfy the needs of clinical detection such as basic hospital, rural hospital, and reduced cost greatly, made things convenient for the patient.The present invention simultaneously diagnoses the degree of accuracy height, thereby makes the patient can access treatment timely, has reached the purpose of control.
Embodiment
Embodiment 1:
The preparation of recombinant antigen
(1) from the body Karp pnca gene group of yochubio east, amplify the big fragment of 1053bp in the sta56 gene ORF, with the TA clone technology with this large dna fragment cloning;
(2) be template with this recombinant plasmid, amplify the fragment of 957bp, directed pET30a carrier, transformed into escherichia coli BL21 (DE3), the 0.2mMIPTG abduction delivering of inserting; Western blot confirms that this recombinant protein can be discerned by yochubio patient positive serum;
(3) electroelution method purification of recombinant proteins: after the thalline of collecting when inducing in a large number carries out Ultrasonic Pulverization, will reset and add sample on 6 * sample loading buffer on ultrasonic, first 80V electrophoresis after edge behind the sample enters separation gel, rises to 110-120V, and electrophoresis spends the night.Electrophoresis is complete to unload glue, uses 0.3mol/L CuCl after the distilled water rinsing 2Dye to destination protein band high-visible (background is light blue, and protein band is the plaque shape) and carefully downcut the destination protein band.The blob of viscose that will contain destination protein place eluent (250mM EDTA, 250mM Tris-HCl, pH9.0) in to remove Cu 2+, incubation is 10 minutes under the room temperature, repeats twice behind the replacing eluent.Blob of viscose is encapsulated in the bag filter, and adds 30ml electrophoretic buffer (being the recombinant antigen lysate), put electrophoresis in the horizontal strip electrophoresis groove, 80V, after the 50min electrophoresis finishes, liquid in the sucking-off bag filter, about 30ml.Purification of samples carries out SDS-PAGE, determines purity and carries out protein quantification.
Embodiment 2:
The making of fast diagnose test paper
(1) get 3/0,000 tetra chlorauric acid 1000ml, be heated to boiling, add 1% citric acid three sodium solution 25ml, continued to boil 5 minutes, treat that the colloidal gold solution color is blue after purple stain is red by having, standby behind the natural cooling;
(2) get colloidal gold solution 1ml, add 0.2M K 2CO 310 μ l, 50 μ g purifying antigens leave standstill 1h after stirring evenly;
(3) add 20% bovine serum albumin(BSA), 25 μ l, 20% PEG, 20,000 15 μ l, 8,000r/min, centrifugal 8min abandons supernatant;
(4) precipitation adds the damping fluid suspension of 1ml basis, is gold mark antigenic solution;
(5) with method mark mouse IgG;
(6) two kinds of gold mark antigenic solutions mix fully sized glass fibres paper of back, put the freeze dryer freeze-drying;
(7) sheep anti-mouse igg monoclonal antibody 3.0mg/ml, goat anti-human igg 3.0mg/ml, goat-anti people μ chain 1.0mg/ml wrap by the NC film in the position, upper, middle and lower of NC film respectively, dry naturally; Or sheep anti-mouse igg monoclonal antibody 3.0mg/ml, recombinant antigen solution 2mg/ml at upper and lower 1/3rd places of NC film bag by the NC film, dry naturally;
(8) on the wide base plate of about 10cm, with thieving paper, NC film, adsorbed the glass fibre of golden mark antigen and not the glass fibre of adsorption antigen be assembled into the test strips that detects IgM and IgG and detect total Ig.
Estimate: on the glass fibre of test strips, add 3 μ l (detecting IgM and IgG) or 5 μ l serum to be checked (detecting total Ig), drip 1～2 reactant liquor again.Observe gold mark antigen and discharge the response situation of detection line and control line on the NC film of back, detection line and control line occur red stripes simultaneously and are judged to the positive, and only control line red stripes occurs and is judged to feminine gender, and it is invalid test that band does not appear in control line.The susceptibility that the gold-marking immunity chromatography detects IgM, IgG, total Ig is respectively 92.3%, 90.9% and 98.6%; Specificity is respectively 94.7%, 94.7% and 93.3%.

Claims (3)

1. one kind is used for the recombinant antigen of yochubio diagnosis and the formulations prepared from solutions of fast diagnose test paper thereof, it is characterized in that:
Solution formula is:
(1) recombinant antigen lysate: SDS 0.1%
Glycocoll 250mmol/L
Tris.Cl 25mmol/L
(2) colloidal gold solution: 2～5/0,000 tetra chlorauric acid adds 1～2% trisodium citrate and is reduced into medium sized colloid gold particle;
(3) two is anti-: anti-human IgG, anti-people IgM resist more, available from Sigma company;
(4) bag is cushioned liquid: 1～3% sucrose in PBS;
(5) basic damping fluid: available from Xiamen Bo Sheng Bioisystech Co., Ltd;
(6) reaction buffer: 0.01～0.05%NaN 3In PBS;
(7) PBS: dissolving 8g NaCl, 0.2g KCl, 1.44g Na in 800ml distilled water 2HPO 4With 0.24g KH 2PO 4, pH value to 7.2～7.6 with hydrochloric acid conditioning solution add water and are settled to 1L.
2. preparation technology's flow process that is used for the recombinant antigen of yochubio diagnosis is characterized in that:
The technological process of recombinant antigen is:
(1) from the body Karp pnca gene group of yochubio east, amplifies sta56 gene ORF or big fragment wherein, use the TA clone technology this large dna fragment cloning;
(2) be template with this recombinant plasmid, amplify the sta56 fragment of brachymemma, directed pET30a carrier, transformed into escherichia coli BL21 (DE3), the IPTG abduction delivering of inserting; Western blot confirms that this recombinant protein can be discerned by yochubio patient positive serum;
(3) adopt electroelution method purification of recombinant proteins: after the thalline of collecting when inducing in a large number carries out Ultrasonic Pulverization, ultrasonic supernatant is prepared electrophoresis, determine the position of target stripe in gel, downcut the gel glue that contains target protein and place bag filter to carry out electroelution; Purification of samples carries out SDS-PAGE, determines purity and carries out protein quantification.
3. fast diagnose test paper that is used for the recombinant antigen of yochubio diagnosis is characterized in that:
The test paper manufacture craft is:
(1) with colloid gold label recombinant antigen and mouse IgG;
(2) two kinds of gold mark antigenic solutions mix fully sized glass fibres paper of back, put the freeze dryer freeze-drying;
(3) goat-anti people μ chain 1.0～5.0mg/ml, goat anti-human igg 1.0～5.0mg/ml and sheep anti-mouse igg monoclonal antibody 1.0～5.0mg/ml bag is dried naturally by the NC film;
(4) with thieving paper, NC film, absorption and not the glass fibre of ADSORPTION OF GOLD mark antigen be assembled into and detect IgM, IgG and total Ig test strips.
CNA031473156A 2003-07-05 2003-07-05 Recombination antigen for tsutsugamushi disease diagnosis and test paper for its rapid diagnosis Pending CN1479099A (en)

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Application Number Priority Date Filing Date Title
CNA031473156A CN1479099A (en) 2003-07-05 2003-07-05 Recombination antigen for tsutsugamushi disease diagnosis and test paper for its rapid diagnosis

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CN1479099A true CN1479099A (en) 2004-03-03

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101253407B (en) * 2005-06-27 2011-11-09 比纳克斯公司 Organic non-sugar compounds for protection of biologically active molecules and conjugate labels and methods thereof
CN102590500A (en) * 2012-01-16 2012-07-18 宁波天润生物药业有限公司 WFR (Weil Felix Reaction) test card
CN108474797A (en) * 2015-12-30 2018-08-31 仁荷大学校产学协力团 Interferon by detecting peripheral blood mononuclear cells is secreted come the diagnostic method of diagnosis of tsutsugamushi disease

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101253407B (en) * 2005-06-27 2011-11-09 比纳克斯公司 Organic non-sugar compounds for protection of biologically active molecules and conjugate labels and methods thereof
CN102590500A (en) * 2012-01-16 2012-07-18 宁波天润生物药业有限公司 WFR (Weil Felix Reaction) test card
CN108474797A (en) * 2015-12-30 2018-08-31 仁荷大学校产学协力团 Interferon by detecting peripheral blood mononuclear cells is secreted come the diagnostic method of diagnosis of tsutsugamushi disease

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